This Phase II proposal is to extend the information gained during Phase I on the validation of a new gas chromatography/mass spectrometry red blood cell folate assay. Using study subjects highly screened for normalcy, a normal reference range has been established; the precision of the assay verified; and optimal storage conditions determined. Using this information a population normal range will be established and a population of human who have a high incidence of folate deficiency will be studied during Phase II. The precision of the gas chromatography/mass spectrometry assay will be tested again, but using samples with a low folate content. After initial sample collection, subjects at high risk for folate deficiency will be treated with folate and observed in the hospital for a minimum of 14 days. Morphologic changes in blood cells that are very specific (but less sensitive) for deficiency will be studied to determine clinical response, thus, confirming folate deficiency. The gas chromatography/mass spectrometry assay method will be compared to standard folate assays on initial samples to determine which assay better predicts for a clinical response to folate. PROPOSED COMMERCIAL APPLICATION: Over tissue folate deficiency leads to anemia and subclinical deficiency is associated with a variety of diseases. Currently all commercial folate assays suffer from difficulties with precision and are poor in the detection of folate deficiency. An accurate folate assay is therefore commercially viable since clinical and subclinical folate deficiency occurs in 20-40% of the US population.